Outline

Introduction: In our cellular model of oral-esophageal carcinogenesis immortalization was induced independent of the activation of telomerase but through alternative lengthening of telomeres (ALT). In this stepwise model additional overexpression of EGFR in immortalized cyclin D1 overexpressing/p53 inactivated cells (OKF6 D1/del.p53) led to in vitro transformation and reactivation of telomerase. Therefore, we sought to identify the cellular pathways involved in the reactivation of telomerase and the in vitro transformation induced by EGFR.

Methods: We overexpressed EGFR in immortalized OKF6 D1/del.p53 cells by retroviral mediated gene transfer. Expression levels and phosphorylation status of the PI3K/AKT and MAPK/ERK pathways as well as respective downstream targets were analyzed by western blot and RT-PCR. Protein-protein-interactions were demonstrated by co-immunoprecipitation. Cells were additionally treated with respective inhibitors and analyzed for growth characteristics and telomere biology. Furthermore, analysis of the hTert promoter including site-directed-mutagenesis was performed.

Results: EGFR overexpression induced a robust telomerase activity in immortalized squamous epithelial cells. The level of AKT expression was unchanged compared to OKF6 D1/del.p53 cells. Nevertheless, AKT was phosphorylated at Ser 473 and Thr 308 indicating the activation of the PI3K/AKT pathway. In contrast, ERK 1/2 levels and phosphorylation status remained unchanged. IP-western blot analysis showed a direct phosphorylation of hTert as well as Hif-1alpha by AKT. Analysis of the hTert promoter indicated an additional transcriptional regulation of telomerase reactivation depending on the presence of a Hif-1alpha binding site at the hTert core promoter. Corresponding to these findings, protein levels of Hif-1alpha were increased in EGFR overexpressing cells.

Conclusion: We were able to show that EGFR reactivates telomerase in immortalized squamous epithelial cells mediated through the PI3K/AKT pathway. This reactivation is effected by two different mechanisms, namely direct phosphorylation of hTert by AKT as well as transcriptional regulation by Hif-1alpha as key transcription factor. Surprisingly, Hif-1alpha is also phosphorylated by AKT and thereby stabilized. Whereas immortalization is induced by ALT, in vitro transformation is dependent on telomerase reactivation, indicating an additional role of telomerase in tumor progression besides elongating telomeres.